Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme-template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme-template complex to the same extent as by an RNA-synthesizing enzyme-template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.     

Identification of heterotrimeric G proteins in human sperm tail membranes

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α-subunits, and A 86, which detects all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein α-subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa β₁-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive α-subunits. However, membrane preparations of nonpurified human spermatozoa contained α₂ subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α-subunits; the putative β-subunit was identified as a β₂-subunit. © 1995 Wiley-Liss, Inc.     

Separation of human X and Y spermatozoa by free-flow electrophoresis

Free-flow electrophoresis is a fast and promising method for gamete separation. Pretreated seminal plasma-free human spermatozoa were injected continuously as a fine stream into the buffer medium of the separation chamber flowing perpendicular to the forces of an electrical field, which separated the spermatozoa according to their differences in electrophoretic mobility. For characterization of the two classes of spermatozoa before and after separation, quinacrine mustard staining was used to identify the Y-chromosome-bearing spermatozoa carrying the fluorescent body (F-body). Human spermatozoa moved toward the anode and were separated into two main peaks. The faster moving fraction contained nearly 80% Y-bearing spermatozoa and the slower peak consisted mainly of pure X-bearing spermatozoa. Whereas sperm viability as determined by eosin staining was nearly unchanged, sperm motility was reduced after free-flow electrophoresis.     

In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotide strategies have been employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression. Pharmacologically, 'code blockers' are ideal agents for antitumour and antimicrobial treatments because of their specific mode of action. Here we report the inhibition of duck hepatitis B virus (DHBV) by antisense oligodeoxynucleotides in primary duck hepatocyte cultures in vitro as well as in DHBV-infected Pekin ducks in vivo. The most effective antisense oligodeoxynucleotide was directed against the 5' region of the pre-S gene and resulted in a complete inhibition of viral replication and gene expression in vitro and in vivo. These results demonstrate the application of antisense oligodeoxynucleotides in vivo and exemplify their potential as human antiviral therapeutics.     

Deficiency in Thrombopoietin Induction after Liver Surgery Is Associated with Postoperative Liver Dysfunction

Background and Aims

Thrombopoietin (TPO) has been implicated in the process of liver regeneration and was found to correlate with hepatic function in patients with liver disease. With this investigation we aimed to determine if perioperative TPO levels were associated with postoperative outcome in patients undergoing liver resection.

Methods

Perioperative TPO was analyzed prior to liver resection as well as on the first and fifth postoperative day in 46 colorectal cancer patients with liver metastasis (mCRC) as well as 23 hepatocellular carcinoma patients (HCC). Serum markers of liver function within the first postoperative week were used to define liver dysfunction.

Results

While circulating TPO levels significantly increased one day after liver resection in patients without liver cirrhosis (mCRC) (P < 0.001), patients with underlying liver disease (HCC) failed to significantly induce TPO postoperatively. Accordingly, HCC patients had significantly lower TPO levels on POD1 and 5. Similarly, patients with major resections failed to increase circulating TPO levels. Perioperative dynamics of TPO were found to specifically predict liver dysfunction (AUC: 0.893, P < 0.001) after hepatectomy and remained an independent predictor upon multivariate analysis.

Conclusions

We here demonstrate that perioperative TPO dynamics are associated with postoperative LD. Postoperative TPO levels were found to be lowest in high-risk patients (HCC patients undergoing major resection) but showed an independent predictive value. Thus, a dampened TPO increase after liver resection reflects a poor capacity for hepatic recovery and may help to identify patients who require close monitoring or intervention for potential complications.     

Sperm preparation for ART

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll^® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep^®, Nycodenz, SilSelect^®, PureSperm^® or Isolate^® were developed in order to replace Percoll^®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.     

Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5′ cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5′ terminus and initiation codon were only separated by a few nucleotides suggesting a 5′ end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13–24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.     

Perioperative Non-Invasive Indocyanine Green-Clearance Testing to Predict Postoperative Outcome after Liver Resection

BackgroundPostoperative liver dysfunction may lead to morbidity and mortality after liver resection. Preoperative liver function assessment is critical to identify preexisting liver dysfunction in patients prior to resection. The aim of this study was to evaluate the predictive potential of perioperative indocyanine green (ICG)-clearance testing to prevent postoperative liver dysfunction and morbidity using standardized outcome parameters in a routine Western-clinical-setting.ResultsPreoperative reduced ICG—plasma disappearance rate (PDR) as well as increased ICG—retention rate at 15 min (R15) were able to significantly predict postoperative liver dysfunction (Area under the curve = PDR: 0.716, P = 0.018; R15: 0.719, P = 0.016). Furthermore, PDR <17%/min. or R15 >8%, were able to accurately predict postoperative complications prior to surgery. In addition to this, ICG-clearance on postoperative day 1 comparably predicted postoperative liver dysfunction (Area under the curve = PDR: 0.895; R15: 0.893; both P <0.001), specifically, PDR <10%/min or R15 >20% on postoperative day 1 predicted poor postoperative outcome.ConclusionPDR and R15 may represent useful parameters to distinguish preoperative high and low risk patients in a Western collective as well as on postoperative day 1, to identify patients who require closer monitoring for potential complications.     

Antisense therapy of hepatitis B virus infection

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is interferon-a with an efficacy of only 30–40% in highly selected patients. The discovery of animal viruses closely related to the HBV has contributed to active research on antiviral therapy of chronic hepatitis B. The animal model tested and described in this article are Peking ducks infected with the duck hepatitis B virus (DHBV). Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA. An antisense oligodeoxynucleotide directed against the 5′-region of the preS gene of DHBV inhibited viral replication and gene expression in vitro in primary duck hepatocytes and in vivo in Peking ducks. These results demonstrate the potential clinical use of antisense DNA as antiviral therapeutics.